Transposase activity was thought to be extinct in humans because DNA movement can be deleterious in higher organisms, resulting in genomic instability and perhaps malignancy. However, we isolated a human transposase protein termed Metnase that had histone methylase and non-homologous end-joining (NHEJ) DNA repair activity. It was found to interact with DNA Ligase IV, consistent with its NHEJ repair activity. Metnase also was an endonuclease preferential for supercoiled DNA. We therefore explored Metnase's role in decatenating replicated chromatids. Metnase interacted with Topoisomerase II (Topo II ), the critical decatenating enzyme, and enhanced its activity in DNA decatenation, both in vitro and intracellularly. The nuclease activity within the transposase domain of Metnase was required for full enhancement of Topo II decatenating activity. Metnase improved the rate at which Topo II decatenated DNA, and increased the ability of Topo II to resist the decatenation inhibitor ICRF-193. The finding that Metnase improved Topo II resistance to ICRF-193 stimulated an investigation into whether it could mediate resistance to the clinically relevant Topo II inhibitor etoposide. We found that Metnase prevented inhibition of Topo II decatenation by etoposide in vitro, and mediated cellular resistance to etoposide, promoting proliferation in the presence of etoposide. Metnase also promoted a more rapid clearance of etoposide-induced DSB. Thus, Metnase appeared to mediate resistance to etoposide-induced DNA damage and cell cycle arrest. This is a novel mechanism of etoposide resistance that is unexplored. This application proposes to define the mechanism by which Metnase mediates resistance to etoposide by asking three questions: 1) What are the upstream signals that regulate the ability of Metnase to reduce etoposide DSBs? 2) What is the downstream pathway by which Metnase reduces etoposide DSBs? 3) Do Metnase levels predic clinical resistance to etoposide in human malignancy? PUBLIC HEALTH RELEVANCE: We have isolated a novel protein termed Metnase that helps chromosomes repair breaks and also untangle, thereby allowing them to separate properly during cell division. Because of these actions, Metnase mediates resistance to the cancer drug etoposide. Understanding the mechanism by which Metnase does this would allow the generation of drugs targeting Metnase, which could help improve the response of cancer patients to etoposide. In addition, the levels of Metnase could predict which patient will respond to etoposide.